Temporal Coordination of the Transcription Factor Response to H2O2 stress

Abstract Oxidative stress from excess H 2 O 2 activates transcription factors (TFs) that restore redox balance and repair oxidative damage. Though many TFs are activated by H 2 O 2 , it is unknown whether they are activated at the same H 2 O 2 concentration or time after H 2 O 2 stress. We found TF activation is tightly coordinated over time and dose dependent. We first focused on p53 and FOXO1 and found that in response to low H 2 O 2 , p53 is activated rapidly while FOXO1 remains inactive. In contrast, cells respond to high H 2 O 2 in two temporal phases. In the first phase FOXO1 rapidly shuttles to the nucleus while p53 remains inactive. In the second phase FOXO1 shuts off and p53 levels rise. Other TFs are activated in the first phase with FOXO1 (NF-κB, NFAT1), or the second phase with p53 (NRF2, JUN), but not both. The two phases result in large differences in gene expression. Finally, we provide evidence that 2-Cys peroxiredoxins control which TF are activated and the timing of TF activation.


Introduction
Hydrogen peroxide (H 2 O 2 ) is a reactive oxygen species with a complex role in cellular physiology. H 2 O 2 is produced as a byproduct of cellular respiration and by over 40 enzymes in humans. 1 H 2 O 2 functions as a second messenger, activating signaling pathways that promote proliferation, differentiation, and wound healing. [2][3][4][5][6] Yet at high concentrations, H 2 O 2 is toxic to cells due to the creation of hydroxyl radicals by the Fenton reaction. Hydroxyl radicals cause DNA damage, lipid peroxidation, and the formation of unfolded/aggregated proteins all of which inhibit cell proliferation and can induce cell death 7,8 . Thus, H 2 O 2 levels must be tightly regulated and rapidly cleared when concentrations are too high as elevated levels of H 2 O 2 are thought to be the underlying cause of many human pathologies 9 .
To counter high levels of H 2 O 2 , metazoans activate several transcription factors (TFs) including p53, FOXO, NRF2 and other TFs, which act to restore the redox state of the cell and repair damage caused by oxidative stress 10 . Upon activation by H 2 O 2 , these TFs upregulate hundreds of target genes in diverse cytoprotective processes including cell-cycle arrest, NADPH/GSH production, ROS scavenger enzymes, DNA damage repair, autophagy, and protein quality control [11][12][13][14][15][16][17] . In addition to their cytoprotective role, both p53 and FOXO can induce cell death by upregulating apoptotic genes 18, 19 .
Given the diverse molecular challenges caused by oxidative stress, which TFs are activated, and their order of activation is likely tightly regulated and dependent on H 2 O 2 concentration 20 . Indeed, oxidative stress is often differentiated broadly into either eustress (mild oxidative stress) or distress (toxic oxidative stress) and it is thought that these different levels of stress activate distinct TFs 1,21 . Yet which TFs are activated at low vs. high oxidative stress and the relative timing of TF activation is not known and is essential for understanding how cells combat oxidative stress and how this breaks down in disease.
In yeast the timing of TF activation is tightly controlled and dependent on H 2 O 2 concentration. This is best illustrated by Pap1, a TF activated by H 2 O 2 stress in ssion yeast 22,23 . Pap1 is activated by Tpx1, a 2-Cys peroxiredoxin (PRDX) protein. Tpx1 activates Pap1 through a redox-relay mechanism, where oxidative equivalents stemming from H 2 O 2 are passed from Tpx1 to a cysteine in Pap1. 22 This leads to the formation of an intramolecular disul de bond in Pap1. The disul de bond in Pap1 causes nuclear accumulation of Pap1 and activation of downstream target genes. Pap1 nuclear accumulation occurs rapidly at low levels of H 2 O 2 , yet at higher concentrations there is a delay in Pap1 activation 24,25 . The delay at high H 2 O 2 is due to hyperoxidation of a key cysteine residue in Tpx1, which blocks Tpx1 dependent redox relays. Hyperoxidation of Tpx1 can be reversed by sul redoxin (SRX1 in yeast, SRXN1 in humans), but this takes time, and thus Pap1 activation is delayed until SRX1 repairs hyperoxidized Little is known about the temporal regulation of H 2 O 2 induced TF activation in mammals. Yet the similarities between the oxidative stress response in yeast and mammals suggests that this is likely.
Similar to yeast, there is strong evidence that PRDX dependent redox relays occur in mammals. A knockout model of PRDX1 and PRDX2 in HEK293T cells showed reduced protein disul de bond formation following oxidative stress, and transient disul de bond intermediates were recovered between both PRDX proteins and hundreds of other proteins, suggesting that PRDX-dependent redox relays control the oxidation state of a large body of proteins 27,28 . Inactivation of PRDX proteins by hyperoxidation also occurs in response to high H 2 O 2 levels, suggesting H 2 O 2 concentration dependent signaling. Further evidence supports a role for PRDX proteins in regulating transcription factors. For example, PRDX2 regulates STAT3 by a redox relay resulting in a disul de bond in STAT3 causing oligomer formation and attenuation of transcription 29,30 . PRDX1 can form disul de bonds with FOXO3, which leads to its retention in the cytosol 31,32 . Yet the role of PRDX proteins in regulating the timing of TF activation in response to H 2 O 2 and how the timing of activation is affected by dose is unclear.
In this study we found that the set of TFs activated by H 2 O 2, and their time of activation is dose dependent. We rst focused on FOXO1 and p53 as both are activated by H 2 O 2 and upregulate genes in overlapping pathways including cell-cycle arrest and apoptosis. Using immuno uorescence and timelapse imaging we found that low levels of H 2 O 2 cause an immediate increase in p53 levels while FOXO1 remains inactive in the cytoplasm. At higher H 2 O 2 concentrations there are two temporal phases of activation: in the rst phase FOXO1 is shuttled into the nucleus within 1 hour, while p53 levels are kept low. In the second phase, FOXO1 exits the nucleus which is followed by an increase p53 levels. The duration of the rst phase, where FOXO1 is active and p53 inactive, increases with H 2 O 2 dose.
Furthermore, we found that other TFs are activated either with FOXO1 (NF-κB, NFAT1) or with p53 (NRF2, JUN) but not both, suggesting coordinated regulation of each group of TFs. The difference in TF activation between the two temporal phases is re ected in large differences in gene expression with increases in ribosome, oxidative phosphorylation, and proteasome genes in phase 1 and NRF2 and p53 target genes involved in NADPH, glutathione, and nucleotide production increasing in phase 2. Finally, we found evidence that the peroxiredoxin/sul redoxin system controls which group of transcription factors is activated. The distinct target genes activated in each phase, coupled with the evolutionary conservation of a PRDX control mechanism, suggests that ordering the transcriptional response to H 2 O 2 is critical for properly restoring redox balance.

Results
Mutually exclusive activation of FOXO1 and p53 in response to H 2 O 2 To determine if FOXO1 and p53 are activated at the same level of H 2 O 2 stress, we performed a dose response in MCF7 cells and immunostained for FOXO1 and p53 ve hours after treatment. FOXO1 is regulated by nuclear/cytoplasmic shuttling, so we measured FOXO1 activation by determining the fraction of nuclear FOXO1 in individual cells 33 . For p53 we measured mean nuclear levels as p53 is predominantly localized to the nucleus and increases due to inhibition of proteasomal degradation. 34 At low H 2 O 2 concentrations nuclear FOXO1 levels were unchanged yet p53 levels increased ( Fig. 1A-C, 20-60 µM). At higher H 2 O 2 concentrations we observed two distinct populations of cells: one population with increased p53 levels and cytoplasmic FOXO1, and a second population with predominantly active (nuclear) FOXO1 and low p53 levels ( Fig. 1A-C, 80-100 µM, Figure S1B for activation thresholds). The proportion of cells with nuclear FOXO1 increased with H 2 O 2 concentration, while cells with high p53 levels decreased. At the highest H 2 O 2 dose (200 µM), most cells had active FOXO1, while p53 active cells were comparable to untreated controls. Cells with activation of both FOXO1 and p53 were < 5% in all doses tested, suggesting that activation of FOXO1 and p53 is mutually exclusive in response to H 2 O 2 .
The response to H 2 O 2 is known to depend on the number of cells in an experiment, and indeed we observed the concentration of H 2 O 2 required to activate FOXO1 increased with the number of cells plated ( Figure S1A). Therefore, for all experiments in this study we took care to plate equal numbers of cells in control and treatment groups to properly measure relative activation of each transcription factor. with Neocarzinostatin (NCS), which causes DNA double-strand breaks and upregulates p53, suggesting a control mechanism to actively suppress p53 activation at high doses of H 2 O 2 ( Figure S1C).
Mutually exclusive activation of FOXO1 and p53 in response to H 2 O 2 is not limited to MCF7 cells as we observed the same pattern in MCF10A, A549 and U2OS cell lines ( Figure S1D-F). The oxidative stress inducing agent menadione also induces mutually exclusive activation of FOXO1 and p53 ( Figure S1G).
However, tert-butyl hydroperoxide activated p53 but not FOXO1 suggesting a different mode of activation ( Figure S1H). Menadione induces formation of superoxide radicals which then dismutate to H 2 O 2 , it is possible that mutually exclusive activation of p53 and FOXO1 is speci c to H 2 O 2 . Together these data suggest that mutually exclusive activation of p53 and FOXO1 is not cell-type speci c but does not occur under all forms of oxidative stress. FOXO1 activation precedes p53 activation at high concentration of HO The dynamics of p53 accumulation also differs in response to higher concentrations of H 2 O 2 . In some cells p53 levels oscillate similar to the 50 µM dose, yet often with a higher initial spike in p53 levels ( Fig. 2A). While other cells show large bursts of p53 levels similar to the response to UV irradiation 39 . The proportion of cells with oscillating p53 levels decreased with dose as shown by autocorrelation analysis, and recently observed in retinal pigment epithelial cells 37 ( Figure S2A-D). These differences correlated with cell survival as the p53 levels in cells that died reached higher levels than those that survived ( Figure  S2G and S2H). In addition, autocorrelation analysis revealed oscillations in p53 activation in surviving cells but not in dying cells (Fig. 2G). Dying cells also showed an increase in the duration of FOXO1 activation as compared to surviving cells, in agreement with our previous study 36 ( Figure S2E,F). Together these data show that higher concentrations of H 2 O 2 cause prolonged activation of FOXO1, a delayed yet stronger p53 response, and an increase in cell death.
Additional HO induced transcription factors are activated with either FOXO1 or p53 The activation of FOXO1 and p53 in two distinct temporal phases prompted us to ask whether other transcription factors are activated with FOXO1 in phase 1, or p53 in phase 2 in response to H 2 O 2 . To identify potential transcription factors, we treated MCF7 cells with PBS as a control and two different concentrations of H 2 O 2 (50µM and 75µM), isolated individual nuclei, and performed single-cell Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and gene expression using the 10X genomics single-cell Multiome kit. Unsupervised clustering of the ATAC-seq data identi ed six separate clusters ( Fig. 3A and 3B). Clusters four and ve represented PBS control nuclei, while the other clusters were observed predominantly in H 2 O 2 treated nuclei. Clusters two and three were enriched with FOXO motifs but not p53 motifs and we refer to these clusters together as the FOXO cluster ( Figure S3A). In contrast, nuclei in cluster six were enriched for p53 motifs, but not FOXO motifs ( Figure S3B). Thus, we can capture cells in phase 1 (FOXO1 active, p53 inactive) and phase 2 (p53 active, FOXO1 inactive) by ATAC-seq.
We then focused on other transcription factor motifs that were enriched with either the FOXO or p53 cluster but not both ( Figure S3A and S3B). Within the FOXO cluster, we observed an enrichment in HSF, GRHL1, NF-KB, ZKSCAN1 and NFAT TF motifs ( Fig. 3C 3C). We also measured nuclear HSF1 levels but did not observe a change in concentration in response to H 2 O 2 ( Figure S3D). Thus the enrichment in HSF motifs might be due to upregulation of HSF2/HSF4, or regulation of these factors independent of an increase in nuclear levels as has been described elsewhere 40 . Cells in the FOXO clusters (clusters 2 & 3) did have an increase in the expression of the canonical HSF target genes HSPA1A (log2 fold-change .98, P < 10^-5) and HSPA1B log2 fold-change 1.1, P < 10^-6), consistent with a role of heat shock protein activation during the FOXO phase.
The p53 cluster was enriched for motifs in the AP-1 family of TFs (JUN, FOS, and ATF subfamilies), as well as NRF2 (NFE2L2 gene) ( Fig. 3D and Figure S3B). The AP-1 family have similar binding motifs which might give false positives for activation 41  The role of the Peroxiredoxin/Sul redoxin system in controlling the switch between p53 and FOXO1 activation Next, we investigated the mechanism underlying the switch from active p53 and inactive FOXO1 at low H 2 O 2 concentrations, to active FOXO1 and inactive p53 at high H 2 O 2 concentrations. Since other transcription factors are activated with either FOXO1 or p53, with distinct mechanisms of control, it is likely that the mechanism is upstream of the direct regulators of each transcription factor. A redox relay stemming from a 2-Cys PRDX protein is a plausible mechanism as redox relays can affect multiple proteins and are switched off by hyperoxidation when H 2 O 2 levels cross a particular threshold, as described below.
2-Cys PRDX proteins function as dimers and harbor two key cysteine residues: a peroxidatic cysteine (Cp), whose thiol group is oxidized to sulfenic acid (SOH) by H 2 O 2 , and a resolving cysteine (Cr), which reacts with the sulfenic acid form of Cp in trans, forming a disul de bond between the two monomers 42 (Fig. 4A). This disul de bond can be resolved by the thioredoxin system, or it can participate in a redox relay, where it transfers oxidative equivalents to downstream proteins, altering their function 43 Figure S4A), consistent with a role for hyperoxidation and inactivation of PRDX1 or PRDX2 as key events in activating FOXO1 and repressing p53 activation.
Furthermore, 10 µM Conoidon A resulted in FOXO1 activation but not p53 activation in the absence of H 2 O 2 ( Figure S4A). Taken together, these data suggest that inactivation of PRDX1 and/or PRDX2 results in FOXO1 activation and suppression of p53 activation in response to H 2 O 2 .
We then tested the speci c role of PRDX1, as it forms disul de bonds with FOXO proteins, and knockdown of PRDX1 increased nuclear FOXO3 in response to H 2 O 2 in a previous study 31,32 . We created a PRDX1 knockout line using CRISPR/Cas9 ( Figure S4B). In the absence of PRDX1, there is a substantial increase in FOXO1 active cells and a subsequent decrease in p53 active cells ). To con rm that the effect of J14 was due to inhibition of SRXN1 and not due to off-target effects, we knocked down SRXN1 using shRNA ( Figure S4F). SRXN1 knockdown showed a similar shift from p53 activation to FOXO1 activation ( Figure S4D). In addition, live-cell microscopy revealed that J14 prolonged FOXO1 activation and delayed p53 activation, supporting the role of SRXN1 in shutting of FOXO1 and activation of p53 (Supp Fig. 4E). Prolonged FOXO1 activation by J14 was accompanied by increased cell death.
If hyperoxidation of PRDX1 is required for FOXO1 activation and p53 repression, SRXN1 overexpression should increase the concentration of H 2 O 2 required for FOXO1 activation. To test this, we established a cell line (SRXN1-OE) in which SRXN1 is expressed from the PGK promoter and veri ed that these cells have reduced PRDX1/2 hyperoxidation by western blot ( Figure S4H). A heatmap of differentially expressed genes revealed broad differences in gene expression between J14 and SRXN1-OE samples (Fig. 5A). Gene Set Enrichment Analysis (GSEA) using a list of known p53 and NRF2 target genes veri ed that these genes were enriched in SRXN1-OE cells when compared to J14 Together these data suggest that in phase 1, cells upregulate critical components for protein production (ribosomes, spliceosome), protein quality control (proteasome, heat shock proteins) and oxidative phosphorylation. In contrast during phase 2, cells upregulate genes in DNA repair, the pentose phosphate pathway, nucleotide biosynthesis, NADPH production and glutathione biosynthesis.  (Fig. 6). In the rst TF phase, FOXO, NF-KB and NFAT are activated by shuttling to the nucleus, while p53, NRF2 and JUN remain inactive. In the second phase, FOXO, NF-KB and NFAT switch off and p53, NRF2 and JUN switch on.
There are several possible reasons for ordering the TF response at high concentrations of H 2 O 2 . The two different TF phases might set the order of repair of oxidative damage. During the rst TF phase there is an increase in expression of ribosome, proteasome, and oxidative phosphorylation genes (components of the electron transport chain). Thus, the initial TF phase might be required for degrading or preventing the formation of protein aggregates, while replacing damaged ribosomes and proteins in the electron transport chain (ETC) for protein and energy production. Indeed, hyperoxidized PRDX proteins assemble into high molecular weight ring structures that function as chaperones with holdase activity [47][48][49] . In addition, ribosomal RNA and proteins are damaged by oxidative stress and restoring ribosomes might be a high priority for cells immediately following oxidative stress to replace damaged proteins 50  which sequester FOXO3 in the cytoplasm 31,32 . In addition, AKT, which phosphorylates FOXO proteins causing cytoplasmic sequestration, is inhibited by disul de bond formation between two cysteines in the protein, which leads to its inactivation and nuclear accumulation of FOXO 62,63 . In future studies, it will be interesting to elucidate the exact steps of these redox relays. Transfection was carried out using the protocol from Mirus using the TransIT Transfection Reagent LT1. The SRXN1 overexpression vector was designed and constructed using VectorBuilder (https://en.vectorbuilder.com/). The vector is a lentiviral vector that expresses mCerulean-NLS-P2A-T2A-SRXN1 from the PGK promoter and harbors a blasticidin resistance gene. The NLS (nuclear localization sequence) tagged mCerulean is separated from SRXN1 with the P2A-T2A self-cleavage site so that SRXN1 is independent of the nuclear mCerulean signal. The mCerulean signal allows the veri cation that the construct is expressed in cells. MCF7 cells were infected with the lentivirus, cells were selected using blasticidin and clones were isolated and validated. The PRDX1 knockout line was made by using a CRISPR/Cas9 lentiviral vector from VectorBuilder (hPRDX1[gRNA#1077] ). MCF7 cells were infected with lentivirus and selected using blasticidin. Individual clones were isolated and validated using Western Blot.

Materials and Methods
SRXN1 knockdown cells were made by using plasmid from Vector Builder (pLV[shRNA]-Puro-U6 > hSRXN1[shRNA#1]) to transfect MCF7 cells. Cells were then selected using puromycin and the knockdown was validated using Immuno uorescence.
Immuno uorescence: Cells (1700 cells/well) were plated in glass bottom 96 multi-well plates (CellVis) or polystyrene plates from CellCarrier-96. Cells were allowed to attach for two days and treated at different time points. They were xed with 2% PFA for 10 minutes, permeabilized using 0.1% Triton X-100 in PBS for 10mins, blocked with 2% BSA in PBS and incubated o/n at 4 o C in primary antibodies made with 2% BSA and 0.1% Tween in PBS. The cells were then washed two times with PBS followed by incubation with secondary antibodies at room temperature for 1 hour. The cells were then washed two times in PBS followed by staining with DAPI and imaged in PBS. Images were analyzed Cell Pro ler (McQuin et al., 2018). To obtain cytoplasmic levels of FOXO1, a ring of 3 pixels wide is was drawn around the nuclear mask and mean cytoplasmic FOXO1 was extracted using this mask. All plots were made using MATLAB. Humidity, temperature at 37 o C and 5% CO 2 levels was maintained using the OKO labs incubation system. Analysis was performed by importing BAM les with the Rsubread package and featureCounts program dropping all genes with counts less than 10 across experimental conditions 64 . We then performed differential expression analysis on the dataset using DEseq2 65 . J14, Sul redoxin, and wild-type samples were compared between 50µM hydrogen peroxide and no treatment. A list of genes was generated for each set of replicates from these comparisons using thresholds after effect size shrinkage using the apeglm package 66 .These lists were combined to evaluate overall expression changes across each treatment condition. We also evaluated expression differences that were of opposite direction across the J14 and sul redoxin conditions. We compared differential expression of untreated sul redoxin and J14 samples with their wild-type counterparts to get an estimate of expression differences from these conditions independent of hydrogen peroxide and con rmed these trends using Principal Component Analysis.
Bulk Gene Set Enrichment Analysis: Gene Set Enrichment Analysis was performed to evaluate differences in sul redoxin and J14 samples treated with 50µM hydrogen peroxide using the clusterPro ler R package 67-69 (Yu et. al. 2012, Wu et. al. 2021) and functions from the enrichplot package [Yu G (2022)  Single Cell Sequencing Analysis: Fragment les generated via 10x Genomics were analyzed using the ArchR pipeline 72 . Untreated cells and those treated with 50uM and 75uM hydrogen peroxide were separated into six clusters using an iterative latent semantic indexing algorithm acting on tiles of 500-bp for each cell using the ArchR function IterativeLSI for dimensionality reduction and Seurat's ndClusters with a resolution of 0.14. Peaks were called in each cluster using MACS2 using the addReproduciblePeakSet function in ArchR. The getMarkerFeatures function was used to generate lists of relevant features from each of clusters 2, 3, and 6 with clusters with high numbers of untreated cells used as background groups (clusters 1, 4, and 5).
Each cluster was analyzed to assess the prevalence of relevant transcription factors with JASPAR2020 binding pro les. Motif enrichment was evaluated on the generated features for each cluster for motifs with FDR < 0.01 and |LFC| > 1.3 using the peakAnnoEnrichment function in ArchR. ChromVAR     The two temporal phases of transcription factor activation cause distinct transcriptional changes.

Supplementary Files
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